StructuralBasisoftheOncogenicInteractionofPhosphatase PRL ... · Paula Giménez-Mascarell, a1Iker Oyenarte, Serge Hardy,b2 Tilman Breiderhoff,c,d3 Marchel Stuiver,e Elie Kostantin,b,f

Structural Basis of the Oncogenic Interaction of PhosphatasePRL-1 with the Magnesium Transporter CNNM2*□S �

Received for publication, September 23, 2016, and in revised form, November 28, 2016 Published, JBC Papers in Press, November 29, 2016, DOI 10.1074/jbc.M116.759944

Paula Giménez-Mascarell,a1 Iker Oyenarte,a1 Serge Hardy,b2 Tilman Breiderhoff,c,d3 Marchel Stuiver,e

Elie Kostantin,b,f Tammo Diercks,a Angel L. Pey,g4 June Ereño-Orbea,a María Luz Martínez-Chantar,h

Reham Khalaf-Nazzal,i Felix Claverie-Martin,j5 Dominik Müller,c3,6 Michel L. Tremblay,b,f,k2,7

and X Luis Alfonso Martínez-Cruza8

From the aStructural Biology Unit, Center for Cooperative Research in Biosciences (CIC bioGUNE), Technology Park of Bizkaia,48160 Derio, Bizkaia, Spain, the bRosalind and Morris Goodman Cancer Research Centre, fDepartment of Biochemistry, andkDivision of Experimental Medicine, Department of Medicine, McGill University, Montreal, Quebec H3A 1A3, Canada, thecDepartment of Pediatric Nephrology, Charité Universitäts Medizin, Berlin, 13353 Berlin, Germany, the dBerlin Institute of Health,Berlin, Germany, the eIn-Cell NMR Laboratory, Department of NMR-supported Structural Biology, Leibniz Institute of MolecularPharmacology (FMP Berlin), Robert-Rössle Strasse 10, 13125 Berlin, Germany, the gDepartment of Physical Chemistry, Faculty ofSciences, University of Granada, Av. Fuentenueva s/n, 18071 Granada, Spain, the hMetabolomics Unit, Center for CooperativeResearch in Biosciences (CIC bioGUNE), Technology Park of Bizkaia, 48160 Derio, Bizkaia, Spain, the iDepartment of BiomedicalSciences, An-Najah National University, P. O. Box 7, Nablus, Palestinian Territory, and the jResearch Unit, Nuestra Señora deCandelaria University Hospital, 38010 Santa Cruz de Tenerife, Spain

Edited by Roger J. Colbran

Phosphatases of regenerating liver (PRLs), the most onco-genic of all protein-tyrosine phosphatases (PTPs), play a criticalrole in metastatic progression of cancers. Recent findings estab-lished a new paradigm by uncovering that their association withmagnesium transporters of the cyclin M (CNNM) family causesa rise in intracellular magnesium levels that promote oncogenictransformation. Recently, however, essential roles for regula-tion of the circadian rhythm and reproduction of the CNNMfamily have been highlighted. Here, we describe the crystalstructure of PRL-1 in complex with the Bateman module of

CNNM2 (CNNM2BAT), which consists of two cystathionine�-synthase (CBS) domains (IPR000664) and represents anintracellular regulatory module of the transporter. The struc-ture reveals a heterotetrameric association, consisting of a disc-like homodimer of CNNM2BAT bound to two independentPRL-1 molecules, each one located at opposite tips of the disc.The structure highlights the key role played by Asp-558 at theextended loop of the CBS2 motif of CNNM2 in maintaining theassociation between the two proteins and proves that the inter-action between CNNM2 and PRL-1 occurs via the catalyticdomain of the phosphatase. Our data shed new light on thestructural basis underlying the interaction between PRL phos-phatases and CNNM transporters and provides a hypothesisabout the molecular mechanism by which PRL-1, upon bindingto CNNM2, might increase the intracellular concentration ofMg2� thereby contributing to tumor progression and metasta-sis. The availability of this structure sets the basis for the rationaldesign of compounds modulating PRL-1 and CNNM2 activities.

The phosphatases of regenerating liver (PRLs),9 consisting ofPRL-1, PRL-2, and PRL-3, are members of the PTP family.These three closely related PTPs are small single-domainenzymes of �20 kDa that share about 80% amino acid sequenceidentity (supplemental Fig. S1) (1) and have a prenylationsequence at the C terminus, which seems to guide their local-ization in the endosomal compartment and at the plasma mem-brane (2). The PRLs are highly expressed in the majority ofhuman solid tumors, as well as hematological cancers, and areconsidered the most oncogenic of all PTPs (3, 4).

* This work was supported in part by Departamento de Educación, Universi-dades e Investigación del Gobierno Vasco Grant PI2010-17, Departamentode Industria, Innovación, Comercio y Turismo del Gobierno Vasco GrantsETORTEK IE05-14 and IE07-202], Diputación Foral de Bizkaia Grants 7/13/08/2006/11 and 7/13/08/2005/14, Spanish Ministerio de Ciencia e Inno-vación (MICINN) Grant BFU2010-17857, Spanish Ministry of Economy andCompetitiveness Grant BFU2013-47531-R, and Ministerio de Ciencia eInnovación CONSOLIDER-INGENIO 2010 Program Grant CSD2008-00005(to L. A. M.-C.). The authors declare that they have no conflicts of interestwith the contents of this article.

� This article was selected as one of our Editors’ Picks.□S This article contains supplemental Figs. S1–S5 and Movies S1–S3.The atomic coordinates and structure factors (codes 5LXQ and 5MMZ) have been

deposited in the Protein Data Bank (http://wwpdb.org/).1 Both authors contributed equally to this work.2 Supported by the Canadian Institute for Health Research Operating Grant

343439 (to M. L. T.).3 Supported by Collaborative Research Grant 12.01.134/2bT4 from the Berlin

Institute of Health.4 Supported by Grant P11-CTS-07187 from Junta de Andalucía (Spain).5 Supported by Grant PI14/00760 from Fondo de Investigación Sanitaria,

Spain, co-financed by the European Regional Development Fund, “A Wayto Build Europe.”

6 To whom correspondence may be addressed. E-mail: [email protected].

7 To whom correspondence may be addressed. E-mail: [email protected].

8 To whom correspondence may be addressed: Structural Biology Unit, Cen-ter for Cooperative Research in Biosciences (CIC bioGUNE), Bizkaia Scienceand Technology Park Bldg. 800, 48160-Derio, Bizkaia, Spain. Tel.: 944-061-320; Fax: 944-061-301; E-mail: [email protected].

9 The abbreviations used are: PRL, phosphatase of regenerating liver; CNNM,cyclin M; CBS, cystathionine �-synthase; PTP, protein tyrosine phospha-tase; PDB, Protein Data Bank; ITC, isothermal titration calorimetry; AU,asymmetric unit.

crossmarkTHE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 292, NO. 3, pp. 786 –801, January 20, 2017

© 2017 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.

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PRL-1 was first identified as an immediate early gene inregenerating liver after partial hepatectomy (5), and it hasrecently been shown to be important for liver regeneration (6).Also, increased expression of this PTP has been associated withcell proliferation, invasion, and migration in various cell types(7–10). The oncogenic role for PRL-1 in cancer progression wasfurther strengthened by the observation that its expressionlevel is increased during tumor progression (10 –12) implicat-ing PRL-1 as a potential prognostic marker as well as therapeu-tic target in cancer. PRL-1 crystal structure analysis revealed ashallow active-site pocket that is characteristic of dual specific-ity phosphatase (phosphatases that can act upon tyrosine andserine/threonine residues) (13, 14). Furthermore, this PTPexists as a trimer in the crystalline state, and this could poten-tially regulate its function as well as its membrane localization(7, 13, 14). Interestingly, the active-site residue Cys-104 wasfound to form a disulfide bond with a nearby residue Cys-49,suggesting a potential mechanism for redox regulation (14, 15).Functionally, no direct substrate has yet been identified forPRL-1, but it has been associated with the regulation of severalsignaling pathways (16).

Despite an increasing number of PRL-related studies, theirexact mechanisms of oncogenic transformation or normalphysiological function were not well understood until recently,when a new paradigm was uncovered showing that PRLs regu-late magnesium transport via their binding to magnesiumtransporters of the cyclin M (CNNM) family (17). This associ-ation was independently found by other authors (18), confirm-ing that the association of PRLs with CNNMs causes a rise inintracellular magnesium levels that promote cancer progres-sion (17, 18).

In recent years, the existence of specific genes encoding pro-teins directly involved in the transport of magnesium throughcell membranes has been uncovered (19). This includes thehuman “CBS domain divalent metal cation transport mediator”(CNNM) gene family, also referred to as ancient conserveddomain proteins (20–22), that include four isoforms (CNNM1-4) that are expressed in all human tissues (20) except forCNNM1, which is mainly expressed in the brain (23, 24). Muta-tions in CNNM2 cause familial dominant hypomagnesemia(MIM613882) (23, 25) and have been recently linked to majorneuropsychiatric disorders (26), intellectual disability, and dis-turbed brain development (27). In contrast, mutations inCNNM4, the closest homolog of CNNM2, are considered thecause of the autosome-recessive cone-rod dystrophy withamelogenesis imperfecta (MIM217080) (28, 29). Although it iswell recognized that CNNM family members control intracel-lular magnesium levels, there is a debate about their activity aseither efflux (18) or influx (17) transporters or, alternatively, assensors (23) or homeostatic factors (30) that indirectly regulatetranscellular Mg2� transport. Importantly, recent findingshighlight essential roles of the CNNM family, such as regula-tion of the circadian rhythm (31) and reproduction (32).

Structurally, CNNMs are complex proteins that contain anextracellular N-terminal domain preceding a DUF21 trans-membrane domain (Pfam code PF01595), a “Bateman module”(33–35), and a C-terminal cNMP (cyclic nucleotide monophos-phate) binding domain (Pfam code PF00027) (supplemental

Fig. S2) (36). We recently found that the Bateman module ofCNNM2, consisting of two consecutive CBS domains, associ-ates itself with disc-like dimers commonly referred to as “CBSmodules” (supplemental Fig. S2) that accommodate nucleo-tides and metal ions (37, 38). In the absence of bound nucleo-tide, the CBS module adopts a Y-shaped or “twisted” conforma-tion (PDB code 4IYS) (supplemental Movie S1), where theinterfacial helices H3 and H4 maintain the CBS2 domains fromcomplementary subunits in close contact with each other,although the CBS1 domains are separated (37). Upon binding ofphospho-nucleotides (AMP, ADP, ATP, and MgATP), a net-work of polar interactions centered around Thr-568 is dis-rupted, thus causing a coordinated reorientation of helices H0and H4 (supplemental Fig. S2), concomitant to a relative rota-tion of the two CBS motifs (37). This apparently smooth dis-placement of secondary elements within the subunit induces adramatic structural change in the dimeric disc that evolvestoward a “flat” arrangement (PDB codes 4P1G, 4IY0, and 4P1O)(supplemental Movie S2) in which both CBS1 domains interactsymmetrically (37). Importantly, the conformational change inthe CBS module is believed to be transmitted to the transmem-brane region through the C-terminal �-helix H0 connectingboth Bateman modules with the transmembrane domains, thusputatively modulating the activity of CNNM2 and its ability toregulate Mg2� homeostasis (37). Remarkably, mutation of Thr-568 to isoleucine, as found in patients suffering from familialdominant hypomagnesemia (23), mimics the structural effectof nucleotide binding and stabilizes the flat conformer, thusimpairing the conformational equilibrium of CNNM2, whichbecomes “locked” in a nucleotide-bound-like (non-functional)state (37). The high sequence conservation shared by allCNNMs in the Bateman module regions (supplemental Fig. S1)suggests that a similar behavior might be followed by othermembers of the family.

Interestingly, the Bateman module in CNNM3 mediatesPRL-2 binding via an extended loop from the second CBSdomain (17) (an alignment showing the sequence similarity inthis region across the CNNM members as well as its spatialposition is shown in supplemental Figs. S1 and S2). This loop ishighly conserved in all CNNM family members across species,and phylogenetic analysis revealed that it is present only in spe-cies that contain PRL orthologs (17).

Here we report that, besides phosphonucleotides and metalions, the Bateman module of CNNM2 can associate with eachof the three human PRLs to form stable complexes where eachmolecule is structurally modified. We further describe the crys-tal structure of one of these complexes, PRL-1�CNNM2BAT,both in the absence and in the presence of bound ATP andZn2� ions.

Our data reveal the key residues participating in the interac-tion between these two proteins and show the structural rear-rangements that occur upon complexation, thus providingthe structural basis for the oncogenic association betweenthe PRL-1 phosphatase and CNNM2. Finally, we propose themolecular mechanism by which this partnership raises theintracellular Mg2� content of cancer cells thus contributing totumor growth and the acquisition of oncogenic properties (17).

Structure of PRL-1 in Complex with CNNM2

JANUARY 20, 2017 • VOLUME 292 • NUMBER 3 JOURNAL OF BIOLOGICAL CHEMISTRY 787


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Results

CNNM2 Associates with All Three PRLs—The association ofPRLs with the CNNMs has been demonstrated to control theintracellular magnesium levels, but whether all PRL membershave the same mechanism of action still remains to be eluci-dated. Based on previous findings showing that all PRLs interactwith CNNM3 (17) and CNNM4 (18), and by taking into accountthe close sequence homology (supplemental Fig. S1) and possiblysimilar functions shared by CNNMs, we first assessed whetherCNNM2 interacts with all three PRL members.

In this study, we used the full-length human PRL proteinsand an engineered protein construct, CNNM2BAT (39), com-prising residues 429 –584 and containing the Bateman moduleof CNNM2 and the preceding �-helix (H0) that connect to the

DUF21 transmembrane domain (supplemental Figs. S1 andS2). We found that the three potential PRLs-CNNM2BAT com-plexes are indeed formed (Fig. 1). In apparent contradictionwith former observations describing PRL-1 as a trimer in solu-tion (13), but in agreement with NMR experiments showingthat PRL-3 exists in a monomeric state (40), PRL-1 elutedas a monomer in all our size exclusion chromatography ex-periments. In contrast, pure CNNM2BAT eluted as a dimer(Fig. 1), as we described previously (37). The equivalent PRL-2�CNNM2BAT and PRL-3 CNNM2BAT complexes behavedsimilarly (Fig. 1). Additional experiments co-transfectingV5-tagged full-length CNNM2 and GST-tagged PRL-1, -2, and-3 in HeLa cells, followed by GST pulldown, supported the abil-ity of CNNM2 to bind all PRL members (Fig. 1).

FIGURE 1. CNNM2BAT interacts with all PRLs. A–C, size exclusion chromatography was used to isolate the corresponding PRLs�CNNM2BAT complexes. PurePRL-1, -2, and -3 (peak 1; Mth � 22.6, 21.9, and 22.4 kDa, respectively (where M indicates mass)) eluted as monomers (Ve �17 ml; Mexp �13–15 kDa) when theywere independently loaded into a size exclusion chromatography column. In the absence of PRLs, CNNM2BAT (peak 2; Mth �34 kDa) eluted as a dimer (Ve �16ml; Mexp �25 kDa) (37, 39). The molecular masses of the resulting PRL-1/-2/-3�CNNM2BAT complexes (Mth �40 kDa; marked with black, red and blue asterisks,respectively) were consistent with 2�PRL�2�CNNM2BAT heterotetramers in solution (Ve �14.7; 14.5 and 14.5 ml, respectively; Mexp �43, 48, and 48 kDa,respectively). The calibration trendline is y � �0.2451x � 1.5726 (y � Kav; x � log M). Peak 3 arises from the excess of PRL protein present in the incubationsolution. The corresponding bands in the SDS-polyacrylamide gels are shown on the left of each plot. Bands 1 and 2 correspond to independent chromato-graphic runs of pure CNNM2BAT (Mth/subunit �18 kDa) and PRLs proteins. Mth and Mexp represent the theoretical and experimental molecular mass values,respectively. In agreement with the crystal data, the densitometry analysis (using the ImageJ gel analysis tool; rsb.info.nih.gov) of the two bands obtained inthe wells marked with black, red, and blue asterisks showed a PRL-to-CNNM2BAT ratio of 1.03, 0.97, and 0.95, respectively. The crystals of the PRL-1, -2, and-3�CNNM2BAT complexes shown on the left of each plot, diffracted X-rays to a resolution of 2.4, 7.0, and 11 Å, respectively. D, pulldown experiments ofV5-tagged full-length CNNM2 with GST-tagged PRL-1, -2, or -3 show the ability of CNNM2 to interact with the three PRL members. HeLa cells were transientlyco-transfected with V5-tagged CNNM2 (CNNM2-V5) and GST-tagged PRL-1, -2, or -3 for 24 h. GST pulldowns (PD GST) were immunoblotted with V5 and GSTantibodies. TCL, total cell lysate.




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Structure of the PRL-1�CNNM2BAT Complex—The threePRL�CNNM2BAT complexes could be isolated and crystallized,but only those crystals containing PRL-1 showed suitable prop-erties for a crystallographic analysis (Fig. 1). The lattice param-eters, merging statistics, and the systematic absences obtainedfrom the PRL-1�CNNM2BAT crystals were consistent withspace group I222, with one PRL-1�CNNM2BAT heterodimerper asymmetric unit (AU) (Table 1). The application of thesymmetry operations to the AU revealed the biological unit,a 2�PRL-1�2�CNNM2BAT heterotetramer with elongatedshape that contains one central disc-shaped homodimer ofCNNM2BAT subunits (also known as “CBS module” (34, 35))bound to two independent PRL-1 molecules, each one locatedat opposite tips of the disc (Fig. 2 and supplemental Movie S3).The interaction between the CNNM2BAT subunits (that arerelated by a crystallographic 2-fold axis) occurs through theirinterfacial �-helices (H1, H2, H3, and H4) and buries a surfaceof 1416 Å2, whereas the CNNM2BAT/PRL-1 interface buries491 Å2. The two PRL-1 proteins do not interact with each other.

The association of CNNM2 with PRL-1 induces structuralre-arrangements in both proteins. In CNNM2, the most signif-icant changes affect the extended L553–559 (linking strands �5and �6 (Fig. 2)), which becomes ordered by entering into thecatalytic cavity of PRL-1 (Fig. 3). The exposed and elongated

shape of this loop, as well as the relevance of its location, suggestthat it might be the first anchor point between the two proteinswhen they begin to contact. In support of this, we found that asingle point mutation (D558A) of the critical amino acid ofCNNM2 that enters the catalytic cavity of PRL-1 completelydisrupts PRL-1�CNNM2 complex formation (Fig. 3). In con-trast, the independent substitution of residues located atstrands �4 and �5 of CNNM2 by an alanine, threonine, orasparagine (F526A, D528A/D528N, V551A/V551T, or N553A/N553T) is not sufficient to block the formation of the complex,thus suggesting a secondary role for these residues in the inter-action between the two proteins (Fig. 3 and supplementalMovie S3). Of note, a single substitution of the conserved gly-cine at position 565, located at the middle of �6 (supplementalFig. S1), weakens the protein/protein interaction, probably bycausing a disorientation of strand �6 (Fig. 3) that is transmittedto the preceding region of the polypeptide chain. These dataenhance the key role played by L553–559 and are in agreementwith our recent observations demonstrating that substitutionof Asp-426 in CNNM3 (homolog of Asp-558 in CNNM2)results in losing the ability of this protein to interact with PRL-2(41). Interestingly, the overexpression of the CNNM3 D426Amutant in cancer cells is known to decrease their ability to pro-liferate under magnesium-deprived situation, demonstrating a

TABLE 1Data statistics and refinementOne crystal was used per data set. Values in parentheses are for the highest resolution shell. NA � not applicable.

Proteins PRL-1 � CNNM2BAT PRL-1 � CNNM2BAT � ATP Zn2�

Data collection and processBeamline ALBA, XALOC ESRF ID23.1Radiation wavelength (Å) 0.9795 1.2837Space group/PDB code I222/5MMZ C2/5LXQa (Å) 52.7 166.9b (Å) 128.5 125.6c (Å) 153.9 61.0Molecules per AU 1� (PRL-1 � CNNM2BAT) (1 heterodimer) 2� (PRL-1 � CNNM2BAT) (1 heterotetramer)Resolution (Å) 98.6–2.4 (2.41–2.40) 47.9–3.3 (3.34–3.33)Rsym

a 0.054 (1.151) 0.091 (1.120)Rmeas

b 0.056 (1.197) 0.099 (1.221)Rpim

c 0.016 (0.327) 0.038 (0.478)No. of observations 271,410 119,978No. of unique reflections 20,944 17,868Mean I/�I 26.5 (2.5) 13.9 (1.7)CC1/2 0.99 (0.86) 0.99 (0.63)Completeness (%) 99.8 (86.0) 99.9 (97.9)Redundancy 13.0 (13.5) 6.7 (6.3)Mosaicity (°) 0.2 0.2

Refinement statisticsNo. of working/test reflections 20,921/1019 15,312/1540Rwork

d/Rfreee 0.23/0.25 0.20/0.23

No. of atomsProtein 2299 4648Ligand NA 62 (2 � ATP)/2 (zinc)Water 7 NA

Average B factors (Å2)Protein 78.4 70.7Ligand NA 47.5 (ATP)/81.8 (zinc)Water 73.9 NA

Room mean square deviationsBond lengths (Å)/angles (°) 0.003/0.804 0.001/1.255

Ramachandran plot statistics (%)Residues in most favored regions 97.6 97.3Residues in additional allowed regions 2.4 2.7Residues in disallowed regions 0 0

a Rsym � �hkl �i�Ii(hkl) � I(hkl)�I/�hkl�i Ii(hkl).b Rmeas � �hkl �i�Ii (hkl) � I(hkl)�I/�hkl �i Ii(hkl).c Rpim � Rpim � �hkl �i�Ii (hkl) � I(hkl)I/�hkl �i Ii (hkl).d Rwork � � �Fo � Fc�/�Fo.e Rfree � ��Fo � Fc��Fo, calculated using a random 5% of reflections that were not included throughout refinement.




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PRL-2�CNNM3 complex-dependent oncogenic advantage in amore stringent environment (41). However, the main changesoccurring in PRL-1 are in loops configuring the walls of thecatalytic cavity as follows: the P-loop (residues 103–110), theWPD-loop (residues 68 –77), the TI-loop (residues 137–143),and the �3�2-loop (residues 48 –55) (Fig. 3). PRL-1 shares theHCX5R active site, the P-loop, and the WPD-loop motifs typicalof PTPs (14, 42), and its strong structural similarity to dualspecificity phosphatases (DSPs) suggests that it is able todephosphorylate both tyrosine and serine/threonine residues (14,42). In the complex, and similarly to when substrates or substrateanalogs (e.g. SO4

2�) are bound into the catalytic cavity of PRL-1(13), the P- and WPD-loops collapse toward the cleft and interactwith L553–559 of CNNM2 (Fig. 4). This closed conformation pre-sumably prevents the access of substrates and explains why thephosphatase activity of PRL-1 measured in vitro is significantlyreduced in the presence of CNNM2BAT (Fig. 4).

Interestingly, the conserved Arg-110 at the HCX5R motif ofthe P-loop shifts toward residue Asp-72, as when substrates arebound to the catalytic cavity (Fig. 5). The guanidinium groupatom of Arg-110 forms a salt bridge with Asp-558, which isburied in the catalytic cavity near the substrate site. Thus, res-idue Arg-110 plays an important role by interacting with thecatalytic Asp-72 of the WPD-loop, and it neutralizes the other-

wise electrostatic repulsion that would occur between Asp-72and Asp-558 (CNNM2) upon formation of the complex. Theoverall network of interactions contributes to maintaining theWPD-loop in a substrate-bound-like conformation (Fig. 5).Noteworthy, the presence of residues Gly-73 in the WPD-loopof PRL-1 and Gly-557 in the L553–559 loop of CNNM2,together with a proline at position 559 in CNNM2, allows atighter approximation between the two proteins. Another rel-evant interaction involves hydrophobic residues of the P-loopof PRL-1 (Val-105, Ala-106, Leu-108 and the alkyl chain of Arg-110) and of the L553–559 loop of CNNM2 (Pro-559, Phe-560,and Tyr-561). Residues Ala-140, Pro-7, and Phe-141 of PRL-1(Fig. 3) also contribute to this hydrophobic environment. How-ever, the catalytic cysteine at position 104 forms a sulfur bridgewith Cys-49 (Fig. 3), suggesting that the interaction betweenthese two residues does not play a key structural role in thestability and/or formation of the complex.

The interaction between CNNM2 and PRL-1 is not limited tothe L553–559 loop and the PRL-1 catalytic cavity but involvesadditional elements. The CNNM2-PRL-1 interface involvesresidues from the C-terminal end of strands �4 (Phe-526) and�5, L553–559, and the N terminus of strand �6 of CNNM2.Regarding PRL-1, it involves residues from the last turn of helix�5 (residues 134 and 135), loop �5-�6 (136 –142), and the first

FIGURE 2. Crystal structure of the PRL-1�CNNM2BAT heterotetramer. The structure consists of a flat CBS module (a disc-shaped dimer of CNNM2BAT subunits,in yellow and orange, respectively) (37) associated with two independent PRL-1 molecules (in blue). Loop 553–559, connecting strands �5 and �6, interacts withresidues located at the catalytic cavity of PRL-1. The secondary elements of the second molecule are indicated with asterisks.




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turn of helix �6 (Ser-143 and Lys-144), the central residues ofthe WPD-loop (residues 71–73) and P-loop (residues 106 –198), and in a minor extent the residues configuring the N-ter-minal end (Pro-7) (Fig. 3). Importantly, residues Arg-138 (at thebeginning of loop �5-�6) and Lys-144 of PRL-1 form saltbridges with residues Asp-528 (located in the loop that pre-cedes helix H3 in CNNM2) and Glu-556 (at the tip of L553–559), respectively. Additionally, Arg-138 H-bonds Asn-527.

The alkyl chain of Arg-139 of PRL-1 contributes to the hydro-phobic environment participated in by Tyr-561 and Phe-526 ofCNNM2.

cNMP Domain of CNNM2 Is Not Involved in the PRL-1/CNNM2 Interaction—The participation of apparently fewstructural elements from CNNM2 in the PRL-1�CNNM2BATcomplex, prompted us to investigate the possible implication (ifany) of the C-terminal cNMP domain of the transporter in

FIGURE 3. PRL-1-CNNM2BAT interface. A, left, representation of the molecular surface of CNNM2BAT (yellow) and PRL-1 (blue). The main amino acid residuesinvolved in protein/protein interactions (white frames) are represented with sticks on the right. Of note, Asp-558 of CNNM2 enters into the catalytic cavity ofPRL-1 and occupies the location of substrate analogs (e.g. SO4

2�). This appears to promote the closure of the P- and WPD-loops of PRL-1 (see Fig. 5). Electrondensity evidence for Asp-558 and Arg-110 is shown in the unbiased omit Fo � Fc density map contoured at 3.0�. B, surface representation of PRL-1 showing thefour loops configuring its catalytic cavity (highlighted with an asterisk). The coordinates of PRL-1 are extracted from the PRL-1�CNNM2BAT complex describedin this work. C, single mutation D558A completely abolishes complex formation. HeLa cells were transiently co-transfected for 8 h with GST alone or GST-PRL-1in presence of the full-length CNNM2-V5 (WT) or CNNM2 D558A-V5 mutant (D558A). GST pulldown was performed on cell extracts followed by Westernblotting analysis with either V5 or GST antibodies. D, single point mutation of residues Phe-526, Asp-528, Val-551, or Asn-553 (located at the protein/proteininterface and highlighted in red) by an alanine or threonine is not sufficient to block complex formation. HeLa cells were transiently co-transfected for 24 h withGST alone or GST-PRL-1 in the presence of either the full-length CNNM2-V5 (WT) or various point mutants. GST pulldown was performed on cell extract followedby Western blotting analysis with either V5 or GST antibodies. GFP and GST were used as negative controls. TCL, total cell lysate.




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the interaction between the two proteins. To that goal,two additional constructs containing the cNMP domain only(CNNM2cNMP; residues 593– 875) or the entire intracellularregion (CNNM2BAT-cNMP; residues 429 – 875) were evaluatedin binding assays by means of isothermal calorimetry tech-niques (ITC). As shown in Fig. 6, binding signals were onlyobserved when the CNNM2BAT domain was present in theconstruct, with moderate and similar affinities (KD �0.5 �M)(Table 2). Either for CNNM2BAT or CNNM2BAT-cNMP, thebinding is driven enthalpically (�T�S is virtually zero). Theresemblance in the affinity and entropic/enthalpic signaturesuggests that the binding modes of these constructs to PRL-1are structurally similar. In contrast, binding signals were verysmall or identical to those obtained in the blank titration forCNNM2cNMP. Thus, our data suggest that CNNM2cNMP doesnot participate in the formation of the PRL-1�CNNM2 com-plex, or, alternatively, that the interaction proceeds with verysmall heat exchange (and thus it is not detected well by ITC),which is unlikely because protein/protein interaction oftenleads to significant surface burial (and therefore, bindingenthalpy). Interestingly, our ITC experiments show thatCNNM2cNMP does not interact with CNNM2BAT suggestingthat these two intracellular domains act as independentmodules.

Binding of PRL-1 Flattens the CBS Module of CNNM2—Unexpectedly, the CBS module of CNNM2 adopts a flat discconformation in the complex (Figs. 2 and 7 and supplementalMovie S3) instead of the Y-shaped twisted state (supplementalMovie S1) that one would expect in the absence of bound nucle-otides and/or bound metal ions (37). This flat state is equivalentto that adopted in the presence of bound MgATP (supplemen-tal Movie S2) or in the pathogenic T568I variant protein (37).The flat conformation found in the complex appears to befavored by the presence of electrostatic charges of opposite signthat, due to their strategic distribution along the surface of bothproteins (Fig. 7), exert a mutual attraction that causes the dis-

placement of the CBS1 domain of CNNM2 toward the C-end ofstrand �2 and the N-extreme of helix �1 of PRL-1. Among theresidues involved are Glu-462–Glu-485–Glu-487 and Lys-512–Lys-516 from CNNM2, and His-23–Asn-24 –Asn-27 andGlu-35–Glu-36 –Glu-61 from PRL-1. Our data suggest thatbinding of PRL-1 to the twisted conformation of the CNNM2BAT

dimer causes a concomitant shift of CBS1 domains (Fig. 7) fromcomplementary subunits (37). This rearrangement alters theinteractions between the interfacial helices linking the twoBateman modules in the CBS module, allowing it to progressfrom its twisted toward its flat state (37). Additionally, our dataalso indicate that because CNNM2 interacts with PRL-1 basi-cally through the CBS2 domain, it is likely that the twisted-to-

FIGURE 4. CNNM2BAT decreases the enzymatic activity of PRL-1. 15 �g ofHis-CNNM2BAT and 15 �g of His-PRL-1 were tested either alone or in combi-nation after 30 min of incubation along with a buffer control (CTRL) (0.1mg/ml BSA, 3 mM DTT, 50 mM HEPES, pH 7.0, NaOH). The enzymatic activitieswere evaluated by measuring the rate of hydrolysis of a fluorescent syntheticsubstrate (6,8-difluoro-4-methylumbiliferyl phosphate). To see this enzy-matic activity, 50 �M DiFMUP was used. This experiment was done twice; theresults were pooled (n � 10), and they are represented as mean � 95% con-fidence interval with two-way analysis of variance using CNNM2BAT and PRL-1as two factors (p 0.0001) followed by Tukey’s multiple comparison test withadjusted p values: *, p 0.001; **, p 0.0001.

FIGURE 5. Effect of CNNM2 on PRL-1. A, superimposition of the WPD- andP-loops in apo-PRL-1 (light gray) and SO4-bound PRL-1 (orange). Both struc-tures show the WPD-loop in a closed conformation collapsed toward theSO4-binding position. In contrast, the P-loop appears open and distancedfrom SO4 in apo-PRL-1 but closed (and folded toward SO4) in the SO4-boundcomplex. B, superimposition of the WPD- and P-loops in apo-PRL-1 (light gray)and in the PRL-1�CNNM2BAT complex (light blue). The L553–559 of CNNM2 is inyellow. C, superimposition of the WPD- and P-loops in SO4-bound PRL-1(orange) and in the PRL-1�CNNM2BAT complex (light blue). Binding of CNNM2exerts an effect on both loops that is similar to that of substrate binding.Residue Asp-558 from CNNM2 occupies a position similar to that of SO4.




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flat transition can proceed without altering the relative orien-tation between the two proteins.

Binding of ATP Induces Minor Changes in the PRL-1�CNNM2BAT Complex—The former observations prompted usto explore whether binding of nucleotides and/or of divalentcations induce additional changes in the PRL-1�CNNM2BATcomplex, either promoting or preventing its formation. Basedon the studies by Goytain and Quamme (22) showing thatmurine CNNM2 is a wide substrate selectivity transporter withthe capability to transport Mg2�, Co2�, Mn2�, Sr2�, Ba2�,Cu2�, and Fe2� ion, and on our own structural studies showingthat CNNM2BAT has the ability to bind AMP, ADP, andMgATP (37), we tried to grow crystals of the PRL-1�CNNM2BAT complex in the presence of these potential sub-strates. Buffered solutions containing ATP and Zn2� in acidicmedia yielded crystals of suitable quality for crystallographicstudies. The presence of Zn2� ions and ATP triggers subtlestructural changes in the protein complex that result in aslightly different rearrangement of the molecules within thecrystal, which belongs to the monoclinic C2 space group (unitcell parameters: a � 166.9; b � 125.6; c � 61.0; � � 111.4°), andcontain one 2�PRL-1�2�CNNM2BAT heterotetramer perasymmetric unit (Table 1). In these crystals, the CNNM2BATsubunits (and thus their bound ATP molecules) configuring theCBS module are related by a non-crystallographic 2-fold axis.As we had reported previously for the CBS module of CNNM2alone (37), we found that ATP binds at site S2 of each Batemanmodule in the PRL-1�CNNM2BAT complex (Fig. 8). Interest-ingly, the bound nucleotide barely induces structural changeswith respect to the apo-form, the rearrangements being limitedto a subtle approximation of the CBS1 motifs from complemen-tary CNNM2BAT subunits. These data indicate that substratessuch as MgATP or ZnATP act in the same direction of PRL-1,thus reinforcing the structural change induced by the phospha-tase. Upon completion of the refinement, the structural statis-tics (bond lengths (Å)/angles (°), as well as RW/RF values) werein the acceptable range at this resolution (43) (Table 1).

In former studies, we showed that, at neutral pH and in theabsence of divalent cations, the CNNM2BAT domain catalyzesslow ATP hydrolysis to ADP, where the turnover is mainly lim-ited by weak affinity for the nucleotide (37). We also demon-strated that the barrier to ligand entry is significantly reducedby Mg2� co-binding and that a shorter polyphosphate chain inthe nucleotide (with nominally less negative charge) favorsthe protein/ligand interaction, suggesting that negative charge

FIGURE 6. Binding assays. Titrations of CNNM2 proteins. A, raw titrations ofbuffer (blank), CNNM2BAT (23 �M; residues 429 –584), or CNNM2BAT-cNMP (29�M; residues 429 – 875) with PRL-1 (540 �M). B, raw titrations of CNNM2BATwith CNNM2cNMP (0.78 mM; residues 593– 875). C, raw titrations of PRL-1 withCNNM2cNMP. B and C share the same blank titration obtained after titration ofbuffer with a CNNM2cNMP (0.78 mM) solution.

TABLE 2Thermodynamic binding parameters for the interaction betweenPRL-1 and CNNM2 constructs

Protein N Ka Kd �H �T�S

10�6 (M�1) �M kcal�mol�1 kcal�mol�1

CNNM2BAT 2.02 � 0.03 1.25 � 0.01 0.64 � 0.14 �8.9 � 0.1 0.4

CNNM2BAT-cNMP 1.25 � 0.01 2.0 � 0.3 0.36 � 0.04 �8.2 � 0.1 �0.3




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repulsion between the acidic cluster formed by residues Glu-570, Asp-571, and Glu-574 at site S2 and the nucleotide’spolyphosphate chain might play a major role in inhibitingligand entry (37). These data prompted us to analyze the behav-ior of ATP in solution, both in the presence or in the absence of

the target proteins, to find out whether the hydrolysis phenom-enon observed at pH 7 is reproduced (or not) under the currentcrystallization conditions, pH 4.6. To that aim, we monitoredthe hydrolysis of ATP by CNNM2BAT and by the PRL-1�CNNM2BAT complex, by means of 31P NMR. In a control with-

FIGURE 7. Effect of PRL-1 on CNNM2. A, left, structure of apo-CNNM2BAT (37) (yellow) overlapped with PRL-1�CNNM2BAT (PRL-1, cyan; CNNM2, orange). Right,representation of the molecular surface and electrostatic potential of PRL-1�CNNM2BAT. Positively and negatively charged residues are in blue and red,respectively. Binding of PRL-1 to CNNM2BAT causes a shift (indicated with an arrow) of the CBS1 domain that has a similar effect as that caused by MgATP (37).This structural rearrangement is favored by the electrostatic attraction between residues of opposite charge distributed along the surface of both proteins. B,such modification in each subunit makes the CBS module (dimeric association of two CNNM2BAT subunits) progress from a twisted to a flat conformation (37).




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out CNNM2BAT, we found that ATP remained perfectly stableat pH 7 and 4.6 over at least 24 h, regardless of MgCl2 or ZnCl2addition (supplemental Figs. S3 and S4). In the presence ofCNNM2BAT (50 �M versus 1 mM ATP, pH 7), however, 50% ofthe ATP was hydrolyzed to ADP and free phosphate within 16 h(supplemental Fig. S3). No further processing of either ADP orAMP was observed. In agreement with former studies (37),addition of divalent cations strongly impeded ATP hydrolysis atpH 7. Within the same 16 h, only 20% ATP was hydrolyzed ifMgCl2 (5 mM) was added. Remarkably, Zn2� (added as 5 mM

ZnCl2), but not Mg2�, completely stalled ATP hydrolysis byCNNM2BAT either alone or bound to its partner protein PRL-1(supplemental Fig. S3). Interestingly, the monovalent H3O�

cation effected similar inhibition of ATP hydrolysis, with only20% ATP hydrolyzed in 16 h if the pH was lowered from 7 to 4.6.At this elevated H3O� concentration, addition of MgCl2 (5 mM)had no further inhibiting effect on ATP hydrolysis byCNNM2BAT. The observation that increased cation concentra-tions (including H3O� at low pH) effectively hamper ATP hy-drolysis by CNNM2 could be rationalized by charge compen-sation: In the absence of compensating cations, the high localnegative charge density around two ATP molecules bound atclose distance in the PRL-1�CNNM2BAT heterotetramer mightfavor their partial hydrolysis to ADP with reduced negativecharge. Cations bound in the vicinity would effectively screenthe repulsive negative charges and thus reduce the pressure for

ATP hydroxylation. Our data also show that a cation-specificeffect exists, as Zn2� is a more potent inhibitor than Mg2� orH3O�. Briefly, neither CNNM2BAT nor the PRL-1�CNNM2BATcomplex catalyze ATP hydrolysis to ADP and free phosphate inthe presence of Zn2� and/or in acidic media. Of note, bothconditions are met by the crystallization buffer, thus explainingwhy intact ZnATP molecules (and not ADP or a mix of bothnucleotides) are observed in our crystal structures.

In agreement with our NMR analysis, the unbiased omit Fo �Fc maps resulting from omitting the entire nucleotide or the�-phosphates confirmed the presence of two ATP moleculesper CBS module (Fig. 8). Furthermore, the mFo � DFc differ-ence maps revealed residual density consistent with the pres-ence of one Zn2� ion bound to the �- and �-phosphates of eachATP (Fig. 8). This Zn2� ion (Fig. 8) matches the location ofMg2� detected previously (37) and coincides with the positionof Zn2� atoms determined from anomalous difference mapsobtained from crystals of CNNM2BAT grown in the presenceof ZnCl2 (data not shown). Importantly, its positive chargereduces the electrostatic repulsion that otherwise would existbetween the two neighboring ATP molecules and also betweenthe nucleotide’s phosphate moieties and the acidic clusterformed by residues Glu-570 –Asp-571–Glu-574 at site S2 (37).

Discussion

Discovering the capacity of PRLs to interact with members ofthe CNNM family to increase the intracellular concentration ofMg2� ([Mg2�]i) and to promote tumor growth and metastasishas established an unprecedented link between cancer andMg2� homeostasis (17, 18). It has been observed that PRL-1, animportant player for the regulation of cell cycle progression andproper timing of liver regeneration after partial hepatectomy(6), shows elevated levels in patients with hepatocellular carci-noma, and its ectopic overexpression markedly enhances hep-atocellular carcinoma cell migration and invasion (10). Thus,PRL-1 is a potential prognostic marker as well as an attractivetherapeutic target (10).

Interaction of PRL-1 with CNNM2 Alters the Structure ofBoth Proteins—A fact to note is that in forming the PRL-1�CNNM2BAT complex, both proteins suffer structural modifica-tions, although CNNM2BAT is significantly more affected. Thisparticular module appears to be the unique intracellular regionthat participates in the interaction with the phosphatase, andsuffers a twisted-to-flat conformational change that determinesthe overall architecture of the protein complex. In contrast,PRL-1 barely varies its overall fold with respect to its uncom-plexed state (Fig. 5 and supplemental Fig. S5). Upon binding toCNNM2, the P- and the WPD-loops configuring its catalyticcavity collapse toward the interior of the cleft and adopt a sub-strate-bound-like conformation (Fig. 5). Interestingly, the dis-placement of the WPD-loop observed in PRL-1 is not as drasticas that observed for PRL-3, which shows significant differencesin this region (7, 13).

CNNM2 Binding Decreases PRL-1 Activity—As shown in Fig.5, Asp-558 of CNNM2 occupies the position of substrate ana-logs of PRL-1 (13) in the PRL-1�CNNM2BAT complex. This sub-strate analog-like role played by Asp-558, when entering thecatalytic cavity of PRL-1, not only explains the rearrangement

FIGURE 8. Structure of the PRL-1�CNNM2BAT heterotetramer bound toZnATP. A, two Bateman modules of CNNM2 (CNNM2BAT and CNNM2BAT*,shown in yellow surface and ribbons, respectively) interact with two indepen-dent PRL-1 molecules (in blue surface and ribbons, respectively). One ZnATPmolecule binds at site S2, located in the central cavity of each CNNM2BATsubunit. B, ZnATP is shown in balls-and-stick model with the unbiased omit Fo �Fc density map contoured at 3.5 � (in light green) resulting from omittingZnATP prior to calculation of the map. The interatomic distances between thezinc atoms and the phosphate groups of ATP/ATP* are shown on the right. Cand D show the omit Fo � Fc maps resulting from omitting the �-phosphatemoieties of ATP/ATP* (cyan, contoured at 3.5 �) and Zn/Zn* (blue, contoured at8.0 �) prior to calculation of the maps.




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of the P-loop but also shed lights on the decrease in the alreadyvery weak phosphatase activity of PRL-1 observed in vitro. Yet,it also suggests a reciprocal role of CNNM2 on the regulation ofPRL-1 that leads to cell proliferation likely through the rise ofthe important intracellular cation magnesium concentration.

The superimposition of PRL-1 with the structure of otherphosphatases suggests that, despite being currently consideredas a dual phosphatase, oncogenic PRL-1 is most probably apoorly active phosphoserine and/or threonine phosphatase,and not a phosphotyrosine phosphatase, because bulkier aminoacids (e.g. phosphotyrosines) would likely avoid the interactionwith its intracellular partner CNNM2. The lack of key catalyticresidues in PRL-1 that are crucial in the phosphatase activity ofhighly active phosphatases like PTEN explains the very poor invitro activity observed for PRLs (17). An example is the pres-ence of an alanine (instead of a hydroxyl residue) next to thecatalytic Arg-110 (44). In summary, the data presented heresupport recent findings claiming that the main role of PRLsmight not be to dephosphorylate a variety of substrates but tointeract with CNNMs to control their activity as magnesiumtransporters (17).

In light of our new structures, a new question arises onwhether PRL binding affects and/or determines the affinity ofinteraction between CNNM2 and its ligands, such as Mg-ATP.The ability of PRL-1 to induce structural changes inCNNM2BAT that are similar to those triggered by MgATP sug-gests that a prior binding of PRL-1 to CNNM2 might pre-con-figure site S2 for the subsequent accommodation of MgATP(thus, increasing the affinity of CNNM2 for this nucleotide). Incontrast, one would not expect that a prior binding of MgATPto CNNM2 would increase the affinity of CNNM2 for PRL-1,because MgATP binding to CNNM2 does not significantlyaffect the orientation and/or the structure of L553–559 (themain anchoring point of CNNM2 to PRL-1).

The slight modifications observed in PRL-1 contradict a for-mer hypothesis that significant rearrangements would likelyoccur in PRLs upon association with their physiological sub-strates (14). Previous data has pointed out that the functionalunit of PRL-1 might be a trimer (13). However, our data clearlyshow that a trimeric assembly (13) would be sterically impededin the PRL-1�CNNM2 complex, causing clashes with the sur-rounding CNNM2 molecules. These clashes would occur inde-pendently of the conformational state (twisted or flat) ofCNNM2. Thus, if a functional PRL-1 trimer exists, disassem-bling of this species into its component subunits prior to (orduring) its interaction with CNNM2 is an essential requisite inthe molecular mechanism of Mg2� transport.

PRL-1 Interacting Proteins—Only few PRL-1 interacting pro-teins have been identified so far. Among them are the afore-mentioned CNNM2, CNNM3 (17), and CNNM4 (18) trans-porters, the RhoA inhibitor p115 Rho-GTPase-activatingprotein (RhoGAP) (45), the nuclear transcription factor p53(46), the activating transcription factor-5 (ATF-5 or ATFx), and�-tubulin (16). To our knowledge, the structural information iscurrently limited to the structure of PRL-1 bound to a peptidethat shares high sequence identity with a conserved motif in theSrc homology 3 domain of RhoGAP (45). As shown in Fig. 9,binding of CNNM2 to PRL-1 occurs at a distinct place of the Src

homology 3 domain of RhoGAP (PDB code 3RZ2) (45), sug-gesting a wide versatility of the PRL-1 surface to accommodatedifferent partners.

Mechanism of Mg2� Transport—With the structural dataavailable so far, it is not yet possible to unequivocally assign themode of transport (“on” or “off”), as well as the direction of theflux of ions through the membrane (in3 out (efflux), or out3in (influx)) that corresponds to each of the two known confor-mations (twisted or flat) of the CBS module of CNNM2 (37).The structures of the PRL-1�CNNM2BAT complex allow forattaching a few more pieces of this intriguing puzzle. At low[Mg2�]i, the CBS module of CNNM2 does not bind Mg2�

and/or MgATP and adopts its twisted conformation (37) (panel1, Fig. 10). This structural state should correspond to a “trans-port-off” mode if CNNM2 behaves as a Mg2� “importer” or an“importer regulator” in tumor cells, which is structurally inagreement with the structure-to-mode model that we postu-lated for CNNM2 at low [Mg2�]i in the renal epithelia (37).However, it has been described that low [Mg2�]i, or a higherdemand of this cation in tumor cells, favors the expression of

FIGURE 9. PRL-1 interacting proteins. A, representation of the molecularsurface and electrostatic potential of the interaction face of PRL-1 withCNNM2. Positively and negatively charged residues are in blue and red,respectively. Residues Gly-555, Gly-557, and a Pro-559 in L553–559 providesufficient flexibility to the main chain of CNNM2 to orient residues Glu-554,Glu-556, and Asp-558, toward positively charged areas of PRL-1. Residue Asp-558 from CNNM2 buries into the catalytic cavity of PRL-1 between the WPD-and P-loops. B, surface and ribbon representation of the interacting zone ofPRL-1 (blue) with CNNM2 (yellow) and Src homology 3 domain of RhoGAP(green). As shown, CNNM2 and RhoGAP interact at opposite faces of PRL-1.




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PRLs (17). Thus, as the number of PRL molecules increases, thePRL-1�CNNM2 complex would be formed gradually (panel 2,Fig. 10). Our new structural data suggest that, in the earlystages, the nascent PRL molecules anchor to the L553–559 loopof CNNM2. Subsequently, the complementary charges distrib-uted along the surfaces of both proteins (Fig. 7) would help tostrengthen their interaction making the CBS module to pro-gress from its twisted toward its flat state (Fig. 7). This confor-mational change would be transmitted to the DUF21 trans-membrane domain through helix H0 from each subunit (Fig. 2)(37), thus allowing access of Mg2� ions into the intracellularmedia (panel 3, Fig. 10). Concomitantly, an increased [Mg2�]ifavors its interaction with ATP and enhances the affinity of thelatter for CNNM2 (panel 4, Fig. 10), thus contributing to main-taining the flat conformation and the “transport-on” state whileMg2� consumption is demanded. Keeping in mind that the roleof CNNM2 might differ in different cell types and tissues, itseems reasonable to assume that each of the conformations ofCNNM2 may correspond to a unique state of transport, either“on” or “off,” but not to two of them. Thereby, the twisted andflat conformations are only consistent with an off and an onstate, respectively, if CNNM2 works favoring Mg2� efflux in therenal epithelia (37), although the influx of this cation is in thetumor cell. With the lack of further data, the available struc-tures suggest that CNNM2 might play a dual role, thus allowinga bidirectional flux of Mg2� ions depending on the cell type andtissue.

Structural Basis for Therapeutic Intervention—The pre-sented data lead us to hypothesize that the design of peptideswith an amino acid sequence similar to that of the extendedloop of CNNM2 might be a useful tool to impair the associationof PRL-1 with CNNM2, and it represents a potential strategyfor drug design in cancer treatment. We should note that theparticipation of an extended loop in protein/protein interac-tions is not restricted to the CNNM family and has been for-merly observed in other CBS domain-containing proteins suchas human cystathionine �-synthase, where this structural ele-ment links the last two �-strands of the CBS2 domain and is keyto maintaining the tetrameric assembly of the enzyme (47).Considering the sequence similarity shared by all members

of the PRL family and also across the CNNM proteins, thetherapeutic approach mentioned above might be potentiallyextended to the rest of PRL members. In support of this, wefound that the single substitution of the conserved aspartateresidue located at the tip of the extended loop in at least twomembers of the CNNM family (Asp-558-CNNM2; Asp-426-CNNM3) has similar consequences and completely abolishesthe CNNM/PRL interaction (Fig. 3) (41).

Because magnesium is an essential intracellular cation regu-lating numerous cellular functions, we believe that the elucida-tion of the PRL-1�CNNM2 complex presented here representsan important step to a better understanding of the physiologicalrole of magnesium. In addition, it provides a molecular basis todecipher the function of the PRLs on how their recruitment bythe CNNMs regulates their respective activities, modulatesnucleotide binding, and affect magnesium transport in normalor tumor cells. This first elucidation of a PRL-CNNM complexshould help to understand the relation between magnesiumhomeostasis and cancer.

Experimental Procedures

Expression and Purification of CNNM2BAT, CNNM2cNMP,and CNNM2BAT-cNMP—The CNNM2BAT protein was pro-duced as described previously (39). Use of EDTA was avoided topreclude Mg2� and Zn2� chelation. Fractions containing thepure protein were concentrated to 20 mg ml�1 and were frozenin liquid nitrogen and stored at �80 °C.

The CNNM2cNMP and CNNM2BAT-cNMP protein constructswere generated by recombinant PCR fusing the cDNA encod-ing the intracellular cNMP binding domain (amino acid resi-dues 593-875 and STOP) or the C terminus (Bateman domain �cNMP binding domain; amino acid residues 429 – 875 andSTOP) of murine Cnnm2 to cDNA encoding a His6 tag fol-lowed by a tobacco etch virus protease cleavage site. Theobtained PCR products were cloned into pET22 via BamHI andEcoRI and confirmed by sequencing. Resulting constructs weretransformed into Escherichia coli BL21 (DE3) and used for pro-tein purification. CNNM2cNMP and CNNM2BAT-cNMP purifi-cation involved the use of a nickel affinity column (HisTrap HP,5 ml, GE Healthcare) to isolate the His6 tag fused proteins. A

FIGURE 10. Postulated mechanism of Mg2� transport in tumors is shown (panels 1 and 2). At low [Mg2�]i or if magnesium is required by the cell, PRL-1 (in cyan)expression is increased and anchors to the twisted CBS module (yellow-orange) of CNNM2 (panel 3). Then, the electrostatic attraction between the surfaces ofthe two proteins flattens the CBS module, thus permitting the influx of Mg2� into the cell (panel 4). The effect of PRL-1 on CNNM2 is complemented by aprogressively increased [Mg2�]i, which results in binding of MgATP to the flat CBS module. Note: the structures represented in panels 1, 3, and 4 are extractedfrom the crystal structures of apo-CNNM2BAT (PDB code 4IYS), PRL-1�apo-CNNM2BAT (code 5CJK; 5MMZ is updated code), and PRL-1� ZnATP-CNNM2BAT (code5LXQ), respectively. The structure of the PRL-1�CNNM2BAT(twisted) complex represented in panel 2 is an in silico model created from apo-CNNM2BAT (PDB code4IYS) and apo-PRL-1 (PDB 5MMZ).




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subsequent tobacco etch virus protease cleavage and a secondNi-affinity chromatography step served to eliminate the tagfrom the protein solution. Finally, a molecular size exclusionchromatographic step using a HiLoad 16/600 Superdex 75preparation grade in the case of CNNM2cNMP or a HiLoad16/600 Superdex 200 preparation grade in the case ofCNNM2BAT-cNMP (purchased from GE Healthcare) was per-formed. The isolated pure proteins were aliquoted, flash-frozenin liquid nitrogen, and stored at �80 °C.

Expression and Purification of PRLs—Mouse PRL-1 (a kindgift from Qi Zeng, Singapore) that shares 100% amino acidsequence identity with human PRL-1 was amplified by PCR(KAPA HiFi DNA Polymerase, Kappa Biosystem) using specificprimers for the Gateway system (Invitrogen) and cloned inpDONR221 according to the manufacturer’s instructions. Sub-sequently, it was shuttled into the bacterial expression vectorpDEST17 that contains an N-terminal His6 tag, according tothe Gateway system protocol (Invitrogen). 20-ml starter cul-tures were grown overnight at 37 °C and used to inoculate 2-li-ter cultures of Luria-Bertani medium containing 100 mg liter�1

ampicillin. The cells were grown at 37 °C to an A600 nm of 0.8.Protein expression was induced by the addition of isopropyl�-D-1-thiogalactopyranoside to a final concentration of 1 mM,and the cultures were left shaking for a further 3 h at 37 °C. Thecells were harvested by centrifugation at 11,000 � g for 20 minat 4 °C, resuspended in lysis buffer (50 mM Tris-HCl, pH 8, 300mM NaCl, 25 mM imidazole, 1 mM DTT, 1 mM benzamidine, 0.1mM PMSF), and lysed by sonication in a Labsonic P sonicator(Sartorius; 12 cycles of 15 s at 90% amplitude with 60 s restingon ice between each cycle to prevent sample overheating). Thecell obtained lysate was clarified by ultracentrifugation at250,000 � g for 60 min at 4 °C, and the supernatant was filteredthrough a 0.45-�m filter before being applied onto a pre-equil-ibrated 5-ml HisTrap HP column (GE Healthcare) connectedto an AKTA FPLC system (GE Healthcare) installed within arefrigerated cabinet at 4 °C. The column was then washed with10 column volumes of buffer A (50 mM Tris-HCl, pH 8, 300 mM

NaCl, 25 mM imidazole, 1 mM DTT), and the bound protein waseluted with buffer B (50 mM Tris-HCl, pH 8, 300 mM NaCl, 300mM imidazole, 1 mM DTT) using a linear gradient over 30 col-umn volumes. The fractions containing the protein of interest(confirmed by SDS-PAGE) were pooled and concentratedusing an Amicon Ultra-15 (5000-Da cutoff) centrifugal concen-trator (Millipore) to a volume of �2 ml. Subsequently, the con-centrated protein was applied onto a HiLoad Superdex 75 16/60Prep Grade gel-filtration column (GE Healthcare) pre-equili-brated with 20 mM Tris, pH 8.0, 200 mM NaCl, 1 mM DTT andeluted at a flow rate of 0.3 ml min�1. Fractions containing pureprotein were pooled and concentrated to 10 mg ml�1 using anAmicon Ultra-4 5000-Da cutoff concentrator (Millipore) andwere frozen in liquid nitrogen and stored at �80 °C. HumanPRL-2 and PRL-3 were obtained following a similar protocol tothat used for PRL-1.

Purification of the PRL-1�CNNM2BAT Complex—The pureCNNM2BAT and PRL proteins dissolved in 20 mM Tris, pH 8,200 mM NaCl, 1 mM DTT were incubated together for periodsranging from 20 min to 12 h at two different temperatures (4and 20 °C). A protein mix containing 2.5 mg of PRL-1 and 1.3

mg of CNNM2BAT in a final volume of 0.5 ml of buffer 20 mM

Tris, pH 8.0, 200 mM NaCl, 1 mM DTT (approximate PRL-1/CNNM2BAT molar ratio � 1.5) incubated for 2 h at 4 °C ofPRL-1, was finally selected as the optimal condition to form thecomplex. The protein mix was subsequently subjected to sizeexclusion chromatography analysis (Superdex 200 Increase10/300 GL column, flow rate of 0.5 ml min�1, 4 °C) with bufferconsisting of 20 mM Tris, pH 8, 200 mM NaCl, 1 mM DTT. Thefractions containing the corresponding PRL�CNNM2BAT com-plexes (Fig. 1) were pooled and concentrated to �15 mg ml�1

using an Amicon Ultra-15 (5000-Da cutoff) centrifugal concen-trator (Millipore) and distributed in aliquots of 25 �l that weresubsequently flash-frozen in liquid nitrogen and stored at�80 °C. The identity of all independent proteins as well as ofthose forming the complex was confirmed by mass spectrome-try from the corresponding SDS-polyacrylamide gel bands asdescribed previously (39). The approximate molecular mass ofPRL-1, -2, and -3 and CNNM2BAT and the PRLs�CNNM2BATcomplexes was estimated from a plot representing Kav versuslog Mw (where Kav � (Ve � Vo)/(Vt � Vo); Ve � elution volume;Vt � total column volume, and Vo � void volume), using aSuperdex 200 Increase 10/300 GL column (GE Healthcare) pre-viously calibrated with commercially available molecularweight standards (Bio-Rad, catalogue no. 151-1901).

Plasmid Construction, Transfection and Pulldown Experi-ments—Full-length CNNM2 cDNA (a kind gift from J. G.Hoenderop, Radboud Institute for Molecular Life Sciences, TheNetherlands) was recombined into pDONR221 entry vectorusing specific primers and shuttled into pLenti6-v5 destinationmammalian expressing vector according to the Gateway systemprotocols (Invitrogen). Cloning of GST-PRL-1 was previouslydescribed (17). Point mutations in CNNM2 were introduced bysite-directed mutagenesis using the QuikChange site-directedmutagenesis kit (Stratagene). HeLa cells were cultured inDMEM (HyClone) supplemented with 10% fetal bovine serum(FBS) and 10 �g/ml�1 gentamicin at 37 °C with 5% CO2. Trans-fections in HeLa cells were performed using Lipofectamine2000 (Invitrogen) following the manufacturer’s instructions,and media were replaced 4 h after transfection. GST pulldownswere carried out 8 or 24 h post-transfection as described previ-ously (17).

Crystallization of the PRL-1�CNNM2BAT Complex—Crystalsof the PRL-1�CNNM2BAT complex were obtained in 24 h by thehanging-drop vapor diffusion method at 20 °C in 96- and24-well crystallization plates. Drops consisted of 1 �l of thePRL-1�CNNM2BAT complex solution mixed with 1 �l of pre-cipitant solution (0.1 M sodium acetate, pH 4.6, and 2 M sodiumformate). The protein complex concentration was 15 mg ml�1.To obtain the ZnATP-bound complex, the nucleotide (ATP)was independently added to the protein at a final concentrationof 3.7 mM, resulting in an �1:10 protein-to-nucleotide ratio.ZnCl2 (used as a source of Zn2� ions) was added at a finalconcentration of 5 mM. The crystals, obtained under similarconditions (0.1 M sodium acetate, pH 4.6, and 2 M ammoniumformate at 20 °C) and time (�24 h) to those described for theapo-complex, were flash-cooled in liquid nitrogen immediatelyafter they grew. All datasets used in this work were collected atEuropean Synchrotron Radiation Facility (Grenoble, France)




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beamlines ID23.1 and ID29 and at ALBA Synchrotron (Barce-lona, Spain) beamline MX XALOC-BL13.

X-ray Diffraction Data Collection, Phasing, and Refine-ment—Diffraction intensities were indexed and integratedby using the software XDS and scaled with XSCALE (48).Data were phased by molecular replacement with PHASER(49) using the structures of the truncated CNNM2BAT con-struct (residues 429 –584, PDB code 4IY0 (37) and PRL-1(PDB code 1XM2) (13)) as the initial search models. Thecrystals of apo-PRL-1�CNNM2BAT belong to the I222 spacegroup and contain one heterodimer per asymmetric unit.The symmetry operator that generates the biological hetero-tetramer 2�(apo-PRL-1�CNNM2BAT) from the heterodimeris (�x, �y � 1, z). The crystals of the ZnATP-bound PRL-1�CNNM2BAT complex belong to the C2 space group andcontain one heterotetramer per asymmetric unit. The struc-tures were refined with PHENIX (50) and/or REFMAC5 (51),alternating manual modeling with Coot (52). The actualmodel does not include residues 579 –584 at the C terminusof CNNM2, as well as 23 residues (MSYYHHHHHHLEST-SLYKKAGFT) inserted before the N terminus of PRL-1 bythe expression vector used. It also lacks the first seven N-ter-minal residues of PRL-1, and residues 158 –173 of the phos-phatase. The crystal characteristics and final refinement sta-tistics are summarized in the Table 1. The figures showingthree-dimensional protein structures were prepared withPyMOL and Chimera. Calculation of surfaces was done withthe PISA server (53).

NMR Analysis—For the ATP hydrolysis experiments, weused the unlabeled CNNM2BAT construct and the PRL-1�

CNNM2BAT complex. We then recorded a series of one-dimen-sional 31P NMR spectra (60 ppm spectral width and 3.5 Hzspectral resolution) with inverse gated 1H decoupling (Brf � 2.2kHz at 3.5 ppm) on our 600 MHz BRUKER AVANCE III spec-trometer equipped with a QXI probe. Its low intrinsic 31Pdetection sensitivity required a 1-h FID accumulation per spec-trum for sufficient signal-to-noise ratio, resulting in a 1-h timeresolution to monitor ATP hydrolysis. To optimize the signal-to-noise ratio per time, the total interscan recovery delay �total

should be set to 1.25 � T1, with T1 � longitudinal relaxationtime. For the three 31P signals of ATP and two 31P signals ofADP (1 mM, pH 4.6, 5 mM ZnCl2), we experimentally derivedT1 � 340 – 440 ms, resulting in �total � 425–550 ms. To level outthe slight differences in T1 and thus allow for quantitative anal-ysis of signal intensities, we increased �total to 2 � T1, max � 880ms, resulting in the accumulation of 4096 transients per 31Pspectrum of 1-h duration. The 31P signals of free phosphate,however, has a substantially longer T1 of 7.4 s. To avoid itsincreasing saturation, we applied a � �90° 31P excitation pulseof 285 �s duration at �15 ppm, at the center of the ATP (andADP) signals. This medium power pulse achieves only � �20°residual excitation for the off-resonant phosphate signal (at 0ppm), approaching the Ernst angle for optimal signal-to-noiseas given by cos� � exp(��total/T1) (54). Thus, saturation of thephosphate 31P signal is avoided, however, at the expense ofallowing only for semi-quantitative analysis of its intensity.

Isothermal Titration Calorimetry—ITC experiments wereperformed in an ITC200 microcalorimeter (Malvern) in 20 mM

Tris, 200 mM NaCl, 1 mM DTT, pH 8, and 25 °C.Interaction of PRL-1 with CNNM2BAT and CNNM2BAT-cNMP—

PRL-1 (540 �M) was loaded in the titrating syringe and theCNMM2 proteins (CNNM2BAT, 25 �M; CNNM2BAT-cNMP, at29 �M) (or buffer in blank titrations) in the cell. Titrations wereperformed by stepwise addition of 1 � 0.5 �l followed by 18 –20injections of 1.8 �l, under constant 800 rpm stirring. Heats wereintegrated, and after subtraction of dilution heats (obtainedfrom titrating buffer with PRL-1), the resulting binding iso-therms were analyzed using a single type of independent bind-ing sites model.

Interaction of CNNM2cNMP with PRL-1 and CNNM2BAT—Either CNNM2BAT (23 �M) or PRL-1 (32 �M) was placed in thecell and titrated using CNNM2cNMP (0.78 mM) in the syringe inan ITC200. Buffer was the same as that used in previousexperiments and 25 °C was the experimental temperature.1 � 0.5-�l � 22 � 1.5-�l injections were performed andspaced at 180 s. A blank titration of CNNM2cNMP (0.78 mM)into buffer was performed using identical conditions. Pro-tein concentrations were determined using the extinctioncoefficients for the protein monomers (PRL-1, 24,410; PRL-2,24,410; PRL-3, 25,900; CNNM2BAT, 4470; CNNM2cNMP,13,410; and CNNM2BAT-cNMP 20,860 M�1 cm�1 at 280 nm,respectively).

PDB Accession Codes—The refined models and structurefactors have been deposited in the Protein Data Bank withcodes 5MMZ (apo-PRL-1�CNNM2BAT) and 5LXQ (PRL-1�CNNM2BAT�ZnATP).

Author Contributions—D. M., M. L. T., and L. A. M.-C. conceivedand coordinated the study and wrote the paper. P. G.-M., I. O., T. B.,M. S., and J. E.-O. purified the proteins and performed the crystallo-graphic analysis. S. H. and E. K. cloned and overexpressed the pro-teins, and performed directed mutagenesis experiments, pulldown,and phosphatase activity assays. A. L. P. performed the ITC analysis.L. A. M.-C., I. O., and T. D. performed the NMR experiments;F. C. M., R. K.-N., and M. L. M.-C. contributed to the design andanalyzed the obtained results. All authors reviewed the results andapproved the final version of the manuscript.

Acknowledgments—We thank the staff of BL13 (XALOC) beamline atALBA (Barcelona, Spain) and of ESRF (European Synchrotron Radi-ation Facility, Grenoble, France) beamlines ID14-1, ID23-1, -2, andID29, and Diamond (Didcot, UK) beamline I04 for support duringsynchrotron data collection. We thank Dr. Adriana Rojas for mainte-nance of the in-house X-ray platform and Nekane Merino for valua-ble assistance with the size exclusion chromatography experiments.

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Michel L. Tremblay and Luis Alfonso Martínez-CruzMartínez-Chantar, Reham Khalaf-Nazzal, Felix Claverie-Martin, Dominik Müller,

Stuiver, Elie Kostantin, Tammo Diercks, Angel L. Pey, June Ereño-Orbea, María Luz Paula Giménez-Mascarell, Iker Oyenarte, Serge Hardy, Tilman Breiderhoff, Marchel

Magnesium Transporter CNNM2Structural Basis of the Oncogenic Interaction of Phosphatase PRL-1 with the

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StructuralBasisoftheOncogenicInteractionofPhosphatase PRL ... · Paula Giménez-Mascarell, a1Iker Oyenarte, Serge Hardy,b2 Tilman Breiderhoff,c,d3 Marchel Stuiver,e Elie Kostantin,b,f